(1) Field of Invention
This invention relates to apparatus for growing crystals under microgravity conditions, and more particularly to apparatus having a large number of growth chambers for growing crystals such as protein crystals by the vapor diffusion method under microgravity conditions.
(2) Description of the Prior Art
One of the methods of growing protein crystals is the vapor diffusion method which decreases the solubility and concentrates the molecules to be crystallized by solvent evaporation. For example, the protein to be crystallized is placed in solution in a growth compartment and a solution of precipitating agent is placed in a reservoir about the protein solution. In microgravity experiments, the precipitating solution generally is absorbed in wicking material in the reservoir. Evaporation of the protein proceeds at a rate at least partly dependent upon the vapor pressure difference between the protein droplet solution and the reservoir, and as evaporation progresses the solution becomes supersaturated with protein and crystallization occurs. Nucleation rate and crystal size are highly dependent upon the rate at which critical supersaturation is approached. As noted in Carter et al U.S. Pat. No. 4,886,646, a slow approach results in decreased crystal nucleation rate and a corresponding increase in crystal size and quality.
Protein crystal growth under microgravity conditions results in substantially increased crystal size and quality. The application of the microgravity environment is the subject of several ongoing investigations which aim to increase the size and internal order of protein crystals. Numerous successful applications of the microgravity environment to the growth of high quality protein crystals have been well documented. This is extremely important, since the ability to produce high quality protein crystals has been the limiting step in a number of important macromolecule structural problems. Presently, the field of macromolecular crystallography is undergoing a major technological revolution which permits more efficient and difficult structure determinations. These improvements coupled with the advances in recombinant technologies are providing an increase in the number of structures determined yearly. At the present time, some 1,000 structures have been determined by crystallographic methods. Based on the rate of increase each year, it is estimated that over 10,000 macromolecular structures will have been determined by the year 2000. In view of this, the current demand for high quality crystals will double approximately every two to three years for the next ten years.
Typically, the growth of large, high quality protein crystals using ground-based methods requires numerous crystallization surveys to identify and maximize the proper growth conditions. While advanced concepts in protein crystallization hardwares seek to optimize the growth of protein crystals by elaborate monitoring and dynamic control of a single experiment, these devices have yet to demonstrate superior crystal quality and appear to be many years away from routine use by practicing crystallographers. This points toward the continuing use of simple multiparameter crystallization screens well into the early years of the Space Station. In addition, this type of screening can be very simple, inexpensive and effective.
Vapor diffusion apparatus specially adapted for growing crystals under microgravity conditions is disclosed in Snyder et al. U.S. Pat. No. 5,103,531, wherein a refrigerator-incubator module receives a carrier assembly which fixes a plurality of tray assemblies within the module. Each tray assembly includes a plurality of sealed chambers with a plastic double barrel syringe and a plug for the double tip of the syringe. Protein solution is contained in one barrel and precipitating solution is contained in the other barrel. Ganging mechanisms operate pistons within the barrels of the syringes and the plugs. When activated, the plug is retracted through a chamber having reservoir material including precipitating solution and the protein solution and precipitating solution are extracted from the barrels to form a drop of lower concentration of precipitating agent than precipitating solution in the reservoir material. Water moves by vapor transport from the protein solution drop to the reservoir precipitant solution until vapor pressure equilibrium is achieved. Crystallization starts at or before equilibrium of the vapor pressure. After crystal growth or the end of microgravity conditions, the protein solution with the crystals suspended therein is drawn back into the barrels which are then plugged. This apparatus requires late loading of the proteins and reagents and late access to the flight vehicle. Also, during activation, mixing of solutions may not always occur successfully. Additionally, and most significantly, the design of the apparatus has a limiting effect on the number of crystal growth chambers available for investigation.
In McPherson U.S. Pat. No. 5,096,676, an apparatus is disclosed for growing protein crystals by vapor diffusion techniques, the apparatus comprising a tray having a plurality of equilibrating solution reservoirs including a column or pedestal disposed within each reservoir and having a protein solution receptacle in the top of the column. A cover with an adhesive material seals the top of the tray to seal the reservoirs from atmosphere. Similar apparatus is disclosed in Carter et al. U.S. Pat. No. 5,130,105 wherein a single level vapor diffusing protein crystal growth tray assembly is proposed having a plurality of individual crystal growth chambers. Each chamber has a pedestal including a crystal growth compartment at the upper end into which protein solution is receivable, the pedestals being slidably received through an annular reservoir which contains a wick in which precipitant solution is absorbed. After the solutions are in place, the upper surfaces of the pedestals are moved upwardly to engage a tape sealent to separate the solutions in the growth compartments and the respective reservoirs. When microgravity conditions are encountered, the pedestals are lowered to permit vapor diffusion crystal growth to proceed.